The Tumor-Specific Expression of L1 Retrotransposons Independently Correlates with Time to Relapse in Hormone-Negative Breast Cancer Patients

Background: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. Methods: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. Results: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. Conclusions: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate

Integration of Biomechanical and Biological Characterization in the Development of Porous Poly(Caprolactone)-Based Membranes for Abdominal Wall Hernia Treatment.

AIMS: Synthetic meshes are the long-standing choice for the clinical treatment of abdominal wall hernias: the associated long-term complications have stimulated the development of a new-generation of bio-resorbable prostheses. In this work, polycaprolactone (PCL) porous membranes prepared by solvent casting/porogen leaching of PCL/poly(ethylene glycol) (PEG) blends with different compositions (different PCL/PEG weight ratio and PEG molecular weight) were investigated to be applied in the field. An optimal porous membrane structure was selected based on the evaluation of physicochemical, biomechanical and in-vitro biological properties, compared to a reference commercially available hernia mesh (CMC). FINDINGS: Selected PCL7-2i membranes (derived from PCL/PEG 70/30, PCL: Mw 70,000-90,000 Da; PEG: 35,000 Da) showed suitable pore size for the application, intermediate surface hydrophilicity and biomimetic mechanical properties. In-vitro cell tests performed on PCL7-2i membranes showed their cytocompatibility, high cell growth during 21 days, a reduced production of pro-inflammatory IL-6 respect to CMC and a significant secretion of Collagen Type I. CONCLUSIONS: PCL7-2i membranes showed biomimetic biomechanical properties and in-vitro biological properties similar to or even better than - in the case of anti-inflammatory behavior and collagen production - CMC, a commercially available product, suggesting potentially improved integration in the host tissue

Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair

Mesenchymal stem cells (MSC) have been derived from different cultured human tissues, including bone marrow, adipose tissue, amniotic fluid and umbilical cord blood. Only recently it was suggested that MSC descended from perivascular cells, the latter being defined as CD146+ neuro-glial proteoglycan (NG)2+ platelet-derived growth factor-R\u3b2+ ALP+ CD34- CD45- von Willebrand factor (vWF)- CD144-. Herein we studied the properties of perivascular cells from a novel source, the foetal human umbilical cord (HUC) collected from pre-term newborns. By immunohistochemistry and flow cytometry we show that pre-term/foetal HUCs contain more perivascular cells than their full-term counterparts (2.5%versus 0.15%). Moreover, foetal HUC perivascular cells (HUCPC) express the embryonic cell markers specific embryonic antigen-4, Runx1 and Oct-4 and can be cultured over the long term. To further confirm the MSC identity of these cultured perivascular cells, we also showed their expression at different passages of antigens that typify MSC. The multilineage differentiative capacity of HUCPC into osteogenic, adipogenic and myogenic cell lineages was demonstrated in culture. In the perspective of a therapeutic application in chronic lung disease of pre-term newborns, we demonstrated the in vitro ability of HUCPC to migrate towards an alveolar type II cell line damaged with bleomycin, an anti-cancer agent with known pulmonary toxicity. The secretory profile exhibited by foetal HUCPC in the migration assay suggested a paracrine effect that could be exploited in various clinical conditions including lung disorders

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876

Grafting alters tomato transcriptome and enhances tolerance to an airborne virus infection

Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVYC-to. The analysis identified graft- and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype